Project Summary In my clinical training, I repeatedly saw the devastating effects of vascular disease on patients in the prime of their lives. Vascular diseases such as coronary artery disease (CAD), stroke, arterial dissection, and migraine headache combine to cause over half the death and disability in the United States. To eradicate vascular disease it will be important to develop new treatments that target the arterial cells where the disease begins. My K08- funded research has been to identify these new pathways using human genetic variation as a guide. The loci associated with multiple vascular diseases have recently been identified through genome-wide association studies (GWAS). These loci represent new therapeutic targets, but their biological mechanisms remain largely unexplored. I have identified the mechanism by which a vascular disease associated variant on chromosome 6p24 distally regulates endothelin-1 expression. This regulatory effect of the non-coding variant is vascular tissue specific, and largely seen in endothelial cells. Whether other cells in the blood vessel are responsible for other gene regulatory effects for this locus remains unknown. For this and other vascular disease-associated loci it will be important to characterize the gene regulatory effects in the relevant cell type or cellular subpopulation. New methods in single cell RNA-sequencing all the identification of the full set of cells in the arterial wall. Droplet-based single cell RNA-sequencing allows for analysis of thousands of cells and detection of rare cellular subpopulations. Using this method, I have identified three distinct subpopulations of endothelial cells with transcriptional signatures that suggest functional specialization. This R03 application seeks to extend this finding to human vascular tissue. The goals of this proposal are to determine the best method for single cell RNA-sequencing and to determine the markers of endothelial cell heterogeneity in human vascular tissue. This will provide an atlas of cell types based on transcriptional signature in vascular tissue and serve as a foundational resource for future functional genomics experiments. With a better understanding of the cell types and gene expression signatures in the arterial wall it will be possible to identify new biological pathways for the treatment and prevention of vascular disease.